Rabbit anti vwf antibody

Mice were perfused and fixed with 4% paraformaldehyde at 72 h after reperfusion. After dehydration with 30% sucrose, the brain was frozen and then cut into 12 μm sections (approximately 1.33 mm from the rostral to the bregma). The slices were then washed with PBS, incubated with 0.3% Triton X-100 for 5 min at room temperature, and consequently blocked with 5% fetal bovine serum (BSA) for additional 30 min. Slices were incubated with rabbit anti-HIF-1α antibody (1 : 200, Abcam, Cambridge, London, UK) and rabbit anti-vWF antibody (1 : 100, Abcam, Cambridge, London, UK) at 4°C overnight, consequently probed with Alexa Fluor 488-conjugated donkey anti-rabbit antibody (1 : 400, Abcam, Cambridge, London, UK) and NeuroTrace red (1 : 2000, Molecular Probes; a dye for labeling of neurons based on Nissl stain), and finally visualized under a microscope (OLYMPUS, BX51) using the DP2-BSW software. Three fields from the penumbra zone for each slice were observed using a 40x objective lens.

The left kidneys of recipient mice were dissected following euthanasia and the transplanted islets were evaluated. Three-µm-thick sections were either stained with hematoxylin and eosin (HE) or subjected to immunohistochemistry (for insulin to identify islets, for von Willebrand factor (vWF) to identify vessels, for porcine C-peptide to identify porcine islets, and for mouse C-peptide to identify mouse islets. The primary antibodies used were mouse anti-pig C-peptide (1:200; Cloud-Clone Corp. MAA447Po21, Katy, TX, USA), mouse anti-mouse C-peptide (1:500; Novus Biologicals NBP1-05433, Centennial, CO, USA) and rabbit anti-vWF antibody (1:100; Abcam, Cambridge, UK). After incubation with primary antibody, donkey anti-mouse IgG (H + L) Alexa488 (1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and Cy3-conjugated goat anti-rabbit (1:100; Jackson ImmunoResearch Laboratories, Inc.) were used as secondary antibodies. Nuclear staining was performed using 4′,6-diamidino-2-phenylindole (DAPI). Histological images were obtained using a BZ-X700 microscope (Keyence, Itasca, IL, USA).

Immunofluorescence analyses were performed to characterize the isolated BOECs using specific ECs marker VWF, and to stain for the pVHL (Novus, Oxon, UK). For this purpose, 5 × 10³ BOECs were seeded on sterile 13 mm diameter coverslips (VWR international, Radnor, PA, USA) placed at the bottom of a 24 well-plate, previously coated with collagen. On the next day, cells were washed with PBS and fixed with 3% PFA for 10 min at RT. After two PBS washing steps, samples were incubated with blocking solution (1% goat serum and 1% BSA, in PBS) for 1 h at RT. Then, cells were incubated overnight at 4 °C with rabbit anti-VWF antibody (1:50) (Abcam). Following this, cells were washed thoroughly four times with PBS and incubated for 1 h at RT with goat anti-rabbit IgG (H + L)-Alexa fluor 568 conjugate antibody (1:200) (Thermo Fisher Scientific). Finally, cells were washed with PBS and coverslips were mounted on glass slides using Prolong-DAPI mounting media (Molecular Probes), and 40× confocal images were taken using the fluorescence confocal microscope Sp5 (DMI6000 CS Leica Microsystems, Wetzlar, Germany). Green and blue channels represent VWF and DAPI stains, respectively.